Detection of dna sequences as risk factors for hiv infection

ABSTRACT

A method for identifying a risk factor for diseases, disorders or conditions, such as those caused by human immunodeficiency virus, using the polymerase chain reaction and specific primers. Methods for treating patients having these diseases, disorders or conditions by antimicrobial treatment of the risk factor by combined antiviral and antibacterial treatment or by sustaining or stimulating the subject&#39;s immune system. Methods for screening biological products including red blood cell preparations. Primers and methods for detecting nucleic acids or microbial agents associated with red blood cells, such as those associated with red blood cells in subjects infected with HIV and undergoing antiretroviral therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of U.S. patent application Ser. No.14/851,837, filed Sep. 11, 2015, now U.S. Pat. No. 10,227,665, issuedMar. 12, 2019, which is a Continuation of U.S. patent application Ser.No. 13/752,003, filed Jan. 28, 2013, now U.S. Pat. No. 9,133,525, issuedSep. 15, 2015, which claims benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application 61/716,123, filed Oct. 19, 2012 and toU.S. Provisional Application 61/591,111, filed Jan. 26, 2012, each ofwhich are each expressly incorporated herein by reference.

The subject matter disclosed in U.S. Application Nos. 61/186,610;61/358,282; 61/476,110; 61/476,545; 12/797,286; 13/168,367; 61/591,111;and PCT/US2010/038160 is hereby expressly incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION Field of the Invention

DNA can be amplified from human red blood cell samples by primersdesigned from DNA sequences encoding a bacterial major surface proteinand 16 s ribosomal RNA (16s rRNA). Primer pairs based on DNA sequencesfor the major surface protein 2 (MSP2) of Ehrlichia/Anaplasma canamplify DNA homologous to DNA from human chromosomes 1 and 7 from redblood cell samples. Primers based on DNA sequences encoding 16s rRNAfrom Anaplasma species can amplify DNA from human red blood cells, butnot from nucleated white blood cells. The amplified DNA is contained insamples of red blood cells from HIV infected individuals as well as fromsome healthy individuals of Caucasian or African origin and represents arisk factor for HIV infection. These primers can be employed in methodsfor assessing risk of HIV infection by amplifying DNA from red bloodcell samples.

Description of the Related Art

Chronic HIV infection causes strong immune depression (AIDS) in mostpatients leading to lethal opportunistic infections or cancers. Specificinhibitors of HIV multiplication are currently used for treating HIVinfected patients before they reach the full-blown stage of AIDS. Suchinhibitors act mostly on the reverse transcriptase and protease of HIVto efficiently suppress virus multiplication and reduce virus load to alow level of less than 40 viral RNA copies per ml of blood. Treatmentresults in a partial recovery of the patient's immune system asevidenced by an increase of CD4 lymphocytes and reduction of or lessenedseverity of opportunistic infections. However, this treatment has to begiven without interruption in order to prevent rebound of virusmultiplication and a subsequent reduction in the numbers of CD4lymphocytes. Rebound of viral infection is evidence of a reservoir ofHIV in infected patients that is not accessible to antiviral treatmentand the existence of this reservoir is generally acknowledged. Inaddition to a reservoir of HIV in infected patients, such patients oftencarry other microorganisms that are associated with HIV infection orthat cause opportunistic infections.

Microorganisms associated with HIV infection that are detectable inhuman red blood cells, but not in human leukocytes or other kinds ofnucleated human cells have not been previously characterized. Theidentification and characterization of microorganisms associated withHIV infection is of interest for purposes of assessing risk of HIVinfection or determining the status of an HIV infected patient, forassessing risk or status of opportunistic infections, and to evaluatemodes of treatment for HIV infected subjects.

SUMMARY OF THE INVENTION

The primers designed and discovered by the inventor provide ways topursue these objectives. Three kinds of primers have been developed andstudied by the inventor.

The first kind of primer was designed based on the gene encoding theouter surface protein 2 of Ehrlichia/Anaplasma a genus of rickettsiales,which are known endosymbionts of other cells. These primers amplifiedDNA homologous to segments of DNA from human chromosomes 1 and 7. Theseprimers are described in Appendix 2.

This first kind of primers were initially designed to detect DNAencoding the major surface protein 2 (MSP2) of Ehrlichia/Anaplasmaspecies. However, neither of the two pairs of primers described byAppendix 2 (Primer Pairs 1 and 2) detected at various annealingtemperatures any related microorganism in the biological samplesinvestigated.

Surprisingly, it was discovered that this first kind of primer amplifiedDNA from human red blood cell samples that was highly homologous to DNAsequences on segments of human chromosomes 1 and 7. Primer Pairs 1 and 2amplified DNA by the polymerase chain reaction (“PCR”) that was 100%homologous with human sequences when the primer sequences themselveswere excluded. The amplified DNA was sequenced and the sequences alignedto sequences described for human chromosome 1 (clone RP11-332J14GI:22024579, clone RP11-410C4 GI:17985906, and Build GRCh37.p5 PrimaryAssembly-) and in human chromosome 7 (PAC clone RP4-728H9 GI:3980548;human Build GRCh37.p5, and alternate assembly HuRefSCAF_1103279188381:28934993-35424761). This was not expected since theprimer pairs had been designed to detect genes encoding a bacterial MSP2gene, not human chromosomal sequences. Furthermore, the amplification ofsuch sequences from samples of red blood cells was in itself surprisingsince red blood cells lack a nucleus containing chromosomes. The abilityto amplify DNA homologous to human DNA from red blood cells is evidencethat the target DNA amplified by these primers is present as anextranuclear or cytoplasmic element, such as a plasmid, or is containedin or bound by a microorganism that invades or is otherwise associatedwith red blood cells. This DNA component may be present on a plasmid orotherwise contained in or bound to a microbe associated with red bloodcells. Its presence represents a risk factor for HIV infection orprogression and/or opportunistic infections.

A second kind of primer was designed based on the sequences homologousto human chromosomes 1 and 7 that were amplified by the first kind ofprimers (MSP2 primers). This kind of primer is useful for identifyingthe target DNA homologous to human chromosomes 1 and 7 in a sample, suchas a red blood cell sample. Such primers, including the first type ofMSP2 primers, are used to detect risks of HIV infection, HIVprogression, risks of opportunistic infections, disease prognosis andresponse to drug treatment in subjects where the presence of DNAhomologous to segments human chromosomes 1 and 7 is a risk factor. Thiskind of primer is exemplified in Appendix 3.

A third type of primer was developed based on the genes from Anaplasmaspecies encoding 16s rRNA. Anaplasma is a genus of rickettsiales. Thistype of primer was found to amplify a sequence of 700 bp of ribosomalDNA that was about 85% identical to the corresponding genetic regions ofRickettsia and about 99% identical to the corresponding genetic regionsof Acinetobacter genus. Acinetobacter is a genus of gram negativebacteria within the class of gammaproteobacteria. The homology ofamplified 16s rDNA with Acinetobacter DNA may be coincidental becauseDNA can be amplified from biological samples that pass through a 450 nMfilter unlike classical Acinetobacter.

These primers identify a bacterial agent associated with red blood cellsthat is related to but not identical to known Rickettsia species. Thisbacterial agent has been identified in red blood cells of not only HIVinfected patients but also in some healthy individuals of Caucasian orAfrican origin. This third type of primer is used to detect risks of HIVinfection, HIV progression, risks of opportunistic infections, diseaseprognosis and response to drug treatment in subjects where the presenceof a target containing the amplifiable 16s rRNA or 16s rDNA is a riskfactor. These primers are described in Appendix 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show scans of gel electrophoresis lanes of different PCRamplicons.

DETAILED DESCRIPTION OF THE INVENTION

Amplification of DNA from biological samples, including blood, plasma,and serum samples or samples obtained from cell culture can be performedusing PCR or other nucleic amplification methods known in the art. Thesemethods can be used to amplify or detect target DNA qualitatively orquantitatively to provide a “yes or no” determination of the presence ofthe target sequence or to quantitatively detect an amount of DNAamplified under controlled conditions.

The DNA amplified by the first and second kinds of primers is higherfrequency in red blood cells obtained from patients infected with humanimmunodeficiency virus compared to healthy individuals. This isespecially the case for patients who have undergone or are undergoingantiretroviral therapy. The quantity of DNA amplified by these primersis reduced after long-term treatment of a patient with antibiotics andthe primers were found not to amplify DNA from white blood cells or fromother human cell lines. DNA has also been amplified from the red bloodcells of healthy African subjects not infected with HIV using theseprimers. These results suggest the amplified DNA is derived from anantibiotic sensitive microorganism associated with red blood cells.

Despite the apparent human origin of this DNA, the data herein show thatthe amplified sequences are associated with a transmissible agent andthat the transmissible agent is closely associated with humanimmunodeficiency virus as explained below.

These sequences were easily detected in the DNA of anucleated red bloodcells (RBCs) in 100% (35 out of 35 subjects) HIV-infected African andCaucasian patients and they could not be detected in the DNA ofnucleated cells including in the white blood cell fraction of the samepatients, nor in human DNA from cultured human cells.

These sequences were rarely detected in the red blood cell fraction ofhealthy African subjects and none were detected in the red blood cellsof the healthy Caucasians tested.

Long term antibiotic treatment (e.g., with doxycycline or azithromycin)of HIV-positive patients for more than three months was found todecrease the intensity of the bands amplified using these primerssuggesting that these bands are induced, generated or otherwiseoriginate from an antibiotic sensitive microorganism.

Amplification of DNA from a supernatant of a short term culture of humancell line HL60 with an extract of RBC from an HIV-positive patientprepared by freeze-thawing RBC and then by removing heavy components bya low speed (10 min. at 1,500 g) centrifugation produced strong DNAbands. The intensity of these bands suggests growth and multiplicationof a microorganism that contains DNA amplified by Primer Pairs 1 and 2.

To further explore this effect, new primers were designed that allowamplification of regions of human genomic DNA adjacent to or includingthose amplified by Primer Pairs 1 and 2. These new primers include:

primers “hChr1114179308 S” upstream of, and “hChr1114179853 AS”encompassing one end of the 237 bp amplicon related to chromosome 1(546-bp long amplicon);

primers “hChr7/4292976 S” upstream of, and “hChr7/4294619 AS” downstreamof the 213 bp amplicon related to chromosome 7 (1,643-bp long amplicon);and the primers described by Appendix 3.

These primers amplify DNA not only from the components of RBCs of HIVinfected Caucasian or African patients, but also from the components ofRBCs of healthy African subjects. However, these DNAs are lacking in allor most of HIV-negative Caucasian subjects. These sequences areamplified after antibiotic treatment of their carrier subjectsindicating that the agent generating them is insensitive to antibiotictreatment. As in the case of the MSP2 primers-amplified sequences, thesekinds of primer pairs amplify DNA present in or associated withanucleated RBC and not in white blood cells or human cell lines. It ispossible that such a microorganism identified with these primers differsfrom the carrier of the initial short DNA sequences and will amplify orcause amplification of human genomic DNA sequences in an integrated orunintegrated manner. The primers disclosed herein permit the design ofdiagnostic tests and treatments aimed at reducing the risk of HIVinfection in important segments of the human population in which thisagent appears and can be detected by amplification of these DNAsequences.

The third kind of primers that identify a previously unknown bacterialagent that is associated with human red blood cells and related to, butnot identical to, known Rickettsia species are provided. These primersare derived from the 16S ribosomal DNA sequences of an Anaplasma speciesand amplify a sequence of 700 bp of ribosomal DNA that is about 89%identical to the corresponding regions of the genome of Rickettsia. Thissequence is about 99% identical to the corresponding regions ofAcinetobacter genus DNA. Besides the primers exemplified herein, otherprimers that amplify the same 700 bp of ribosomal DNA or detectablefragments of this sequences may be designed based on this nucleotidesequence. These primers may amplify 20, 30, 50, 100, 200, 300, 400, 500,600 or 700 nucleotides of this sequence. They may comprise shortportions (e.g., 18-30 bp) of the 700 bp sequence and can be designedbased on methods well known in the molecular biological arts. The tablebelow depicts the various kinds of primers.

Specific embodiments of the invention include, but are not limited tothose described below.

An agent that is associated with red blood cells, especially matureanucleated red blood cells, that passes through a 0.45 micron filter.This agent may be sensitive or insensitive to a particular antibiotic.Agents sensitive to azithromycin or to a cyclin antibiotic have beenidentified. This agent contains, induces, excises, or otherwise providesDNA that is amplified by (i) primer pairs 1 (SEQ ID NOS: 3 and 4) or 2(SEQ ID NOS: 5 and 6), (ii) a pair of primers described by Appendix 3(SEQ ID NOS: 7-14 or SEQ ID NOS: 15-23), (iii), the pair of primersdescribed in Appendix 4 (SEQ ID NOS: 24 and 25); or a pair primers thatamplify at least fifteen, twenty, twenty five, thirty, forty, fifty ormore consecutive nucleotides of the same DNA as is amplified by thespecific primers described herein.

The amplified DNA may be 80%, 85%, 90%, 95%, 99%, up to and including100% identical or similar to human DNA, wherein sequence identity isdetermined by BLASTn using the default setting. Preferred parameters fordetermining the “nucleotide identity” when using the BLASTN program(Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages403-410) are: Expect Threshold: 10; Word size: 28; Match Score: 1;Mismatch Score: −2; Gap costs: Linear.

An agent that is associated with red blood cells, passes through a 0.45micron filter, may be sensitive or insensitive to a particularantibiotic, can be detectable in red blood cells of an HIV patient, butnot detectable in the white blood cells of said patient. Such an agentmay become detectable in the red blood cells of an HIV-infected patientwithin the first year after HIV infection or after initiation ofanti-retroviral treatment. Such an agent may appear or be associatedwith the red blood cells of an African subject or European subject whois HIV-negative.

The agent may be a microorganism, such as a bacterium, or a specifickind of bacterium such as Rickettsia or Rickettsia-like bacteria,Ehrlichia or Anaplasma or a component thereof. Such an agent may containa plasmid, episome, or extra chromosomal element comprising humanchromosomal DNA that is amplified by MPS2 gene primers; or that iscontained in or associated with a red blood cell that contains a plasmidor extra chromosomal element comprising human chromosomal DNA that isamplified by MPS2 gene primers.

Isolated red blood cells may contain the agent as described herein aswell as disrupted or lysed red blood cells, such as a supernatantproduced by freezing and thawing red blood cells after removing whiteblood cells and then removing material that pellets by a low speedcentrifugation, e.g., for 10 min. at 1,500 g. The red blood cellsassociated with the agent are detected by amplifying DNA from them using(i) primer pairs 1 (SEQ ID NOS: 3 and 4) or 2 (SEQ ID NOS: 5 and 6),(ii) a pair of primers described by Appendix 3 (SEQ ID NOS: 7-14 or SEQID NOS: 15-23), (iii), the pair of primers described in Appendix 4 (SEQID NOS: 24 and 25); or a pair primers that amplify at least fifteen,twenty, twenty five, thirty, forty, fifty or more consecutivenucleotides of the same DNA as is amplified by the specific primersdescribed herein.

Another aspect of the invention is the DNA amplified from the agent orfrom red blood cells associated with the agent. This DNA is can beproduced using the primer pairs described herein. The DNA that ispresent in a red blood cell may be from an infectious or replicatingagent per se, from a component of an infectious organism present in theanucleated red blood cell, or from DNA that results from exposure of thered blood cell or its precursor cells to an infectious or replicatingagent.

The amplified DNA from a red blood cell may comprise portions of humanchromosome 1 or 7 including the sequences described in Appendix 5 orAppendix 6 or fragments of these sequences comprising 10, 20, 30, 40,50, 100, 200 or more consecutive nucleotides of these sequences.

The DNA according to the invention may be contained or inserted into avector, such as a plasmid or phage vector containing the isolated orpurified amplified DNA. A host cell can be transformed with the isolatedor purified amplified DNA from the agent or from red blood cellsassociated with the agent.

The invention is also directed to a method for detecting an agent asdescribed herein comprising contacting material from anucleated redblood cells of a subject with primer Pair 1, primer Pair 2, or a pair ofprimers selected from the group consisting of those described inAppendix 3 under conditions suitable for amplification of DNA by saidprimers, and detecting said agent when amplified DNA is detected.

The primers used in this method may be selected from the groupconsisting of (i) primer pairs 1 (SEQ ID NOS: 3 and 4) or 2 (SEQ ID NOS:5 and 6), or (ii) a pair of primers described by Appendix 3 (SEQ ID NOS:7-14 or SEQ ID NOS: 15-23) or a pair of primers that amplify at leastfifteen, twenty, twenty five, thirty, forty, fifty or more consecutivenucleotides of the same DNA as is amplified by the specific primersdescribed herein. Alternatively, a set of primers that amplify the sameDNA fragment amplified by the two primers described above may beemployed. These primers may be designed by methods known in the art andeach may comprise 18-30 or more base pairs of the sequence amplified bythe primers above.

A method for detecting an agent as described herein comprising:contacting under conditions suitable for amplification of target DNAmaterial from red blood cells of a subject with a primer and detectingor recovering the amplified DNA, where the primers are described byAppendix 4:

Primer (sense) (SEQ ID NO: 24) 5′-CTG ACG ACA GCC ATG CA Primer(antisense) (SEQ ID NO: 25) 5′-GCA GTG GGG AAT ATT GGA CA.

Alternatively, a set of primers that amplify the same DNA fragmentamplified by the two primers described above may be employed. Theseprimers may be designed by methods known in the art and each maycomprise 18-30 or more base pairs of the sequence amplified by the twoprimers above.

The biological sample used in the method described above or othermethods described herein may be whole blood or a cellular component ofwhole blood, isolated anucleated red blood cells, isolated red bloodcell precursors, such as erythroblasts, bone marrow or spleen cells, orsubcellular fractions thereof, such as cellular lysates, supernatants orsolid materials. Blood plasma or serum or other bodily fluids or tissuesmay also be used as a biological sample for the methods describedherein. Those of skill in the art can select an appropriate biologicalsample for performance of PCR or select the appropriate conditions forproducing an EMS signalized sample based on the disclosures of thepatent applications incorporated by reference above. Representativebiological samples include whole blood, isolated RBCs, subcellularcomponents, extracts, or lysates of RBCs or their precursor cells, bloodplasma or blood serum, spinal fluid, mucosal secretions, urine, saliva,bone marrow, or tissues.

A method for treating or for reducing the severity of a disease,disorder, or condition associated with the agent comprising treating apatient with an agent that reduces the titer of said agent or thatreduces the amount of DNA amplified from a cell associated with it. Thismethod may also comprise treating the patient with one or moreantibiotics, such as azithromycin or a cyclin antibiotic; with one ormore synthetic or natural immunostimulants, active vaccines, passivevaccines, antioxidants or antibiotics. A patient may also undergotreatment sequentially or simultaneously for viruses or othermicroorganisms or agents capable of causing an immunodeficient disease,disorder or condition. Treatment may be therapeutic or prophylactic andcan include the administration of one or more anti-retroviral drugs orother antiretroviral treatments. The patient may be currently undergoingantiretroviral therapy or therapy to eradicate human immunodeficiencyvirus infection and treatment for the coinfecting bacterium initiated.Other modes of or supplemental treatments include treating the patientwith one or more natural immuno stimulants, antioxidants or antibiotics.

The methods described herein may employ samples from subjects orpatients of different geographic origins or racial or geneticbackgrounds. A subject or patient may be HIV-negative, recently (e.g.,less than one year) HIV-positive, a patient who has been HIV-positionfor more than one or two years, an HIV-positive patient who hasundergone or is undergoing anti-retroviral treatments or other kinds ofpatients who are HIV-positive such as those with AIDS or subjects atrisk of becoming HIV-positive, developing AIDS or opportunisticinfections. Patients may be of African origin or may have lived inAfrica and exposed to biological and environmental agents there.Similarly, a patient may be of European or Caucasian origin or may havelived in Europe or America and exposed to biological and environmentalagents there.

The invention is also directed to a method for treating a disease,disorder or condition associated with an agent described hereincomprising contacting red blood cells with a substance that reduces theamount of DNA amplified from a red blood cell using (i) Primer Pairs 1or 2, (ii) primers described by Appendix 3, (iii) or the primersdescribed in Appendix 4 or primer pairs that amplify at least 20consecutive nucleotides of the amplicons amplified by the primer pairsdescribed above. Such a method for treating a disease, disorder orcondition associated with an agent described herein may comprisecontacting red blood cells of a subject with a substance that reducesthe transmission of said agent to the red blood cell; may comprisereplacing the red blood cells in a subject with red blood cells that arenot associated with said agent or by stimulating the development of newred blood cells in said subject; or may comprise treating blood or redblood cells with an agent that that degrades, crosslinks or otherwiseinterferes or inactivates nucleic acids inside of or associated with ared blood cell.

Another aspect of the invention is a method for screening blood for redblood cells from which DNA can be amplified using (i) primer pairs 1(SEQ ID NOS: 3 and 4) or 2 (SEQ ID NOS: 5 and 6), (ii) a pair of primersdescribed by Appendix 3 (SEQ ID NOS: 7-14 or SEQ ID NOS: 15-23), (iii),the pair of primers described in Appendix 4 (SEQ ID NOS: 24 and 25); ora pair primers that amplify at least fifteen, twenty, twenty five,thirty, forty, fifty or more consecutive nucleotides of the same DNA asis amplified by the specific primers described herein. This methodcomprises contacting a sample of blood or red blood cells with thesepairs of primers and detecting amplified DNA and selecting a bloodsample from which DNA was amplified or alternatively selecting a bloodsample from which no DNA was amplified. For example, a blood sample fromwhich amplified DNA is detected may be further evaluated or cultured todetermine the sensitivity of the red blood cells or the agent associatedwith them to antibiotic or other therapeutic treatments. Alternatively,a blood sample in from which no DNA is amplified may be assessed asbeing free of the agent associated with the DNA amplified by theseprimers.

Example 1. Detection of Amplified DNA in Red Blood Cells

Separation of Red Blood Cells

Standard procedures for separating RBCs from buffy coat and otherperipheral blood components are known. Peripheral blood was processed ona Ficoll gradient to separate the buffy coat from red blood cells. Aftersuch separation it was found that DNA extracted from buffy coat cellswas completely negative as determined by PCR using the primers describedabove while the same primers amplified DNA in the fraction containingthe separated red blood cells. While it cannot ruled out that the agentdetected is externally associated with the red blood cell membranes, itwas found that amplified DNA was only detected in a supernatant preparedby a low speed (1,500 g×10 min.) centrifugation to remove the heavycomponents of a red blood cell lysate. This lysate was prepared byrepeated freeze-thawing of red blood cells isolated from the buffy coat,strong shaking by vortex, and a low speed centrifugation (1,500 g×10min.). A pellet and supernatant fraction were obtained and tested. Theprimers described above only amplified DNA in the supernatant fraction,but not in the pellet.

Growth on HL-60 Cells

HL-60 cells are an ATCC cell line of promyelocytic origin. Samples ofHL-60 cells at a density of 5×105 cells per ml in RPMI mediumsupplemented with 10% fetal calfserum were inoculated with thesupernatant of the red blood cell lysate described above. This lysatewas obtained from the red blood cells of HIV-positive patients afterfreezing, thawing and vortexing as previously described. After culturingfor 3 days at 37° C. the low speed (1,500 g×10 mins) supernatants of thecultures were tested by PCR for DNA amplified using Primer Pairs 1 and2. DNA was amplified from all of these cultures up to a dilution of10-8. The same results were obtained from culture supernatant that waspassaged through a 0.45 micron filter.

Effects of Long-Term Antibiotic Treatment

Five HIV-positive patients were maintained on their antiretroviraltherapy, but received for at least three months a daily antibiotictreatment (azithromycin 250 mg/day or doxycycline 100 mg/day). Bloodsamples were fractionated to recover a red blood cell fraction on day 0and after 3 months of antibiotic. Results indicated that the amount ofDNA amplified after 3 months of antibiotic treatment was significantlyless than that amplified under the same conditions from the samplesobtained on day 0.

Detection of Amplified DNA in Red Blood Cells of African and CaucasianPatients

Blood samples were obtained from African and European Patients who wereHIV-negative or HIV-positive. Red blood cells were isolated from buffycoat and other blood components by separation on a Ficoll gradient asdescribed above. Table 1 shows the results of amplification of red bloodcell samples from these patients using Primer Pairs 1 and 2. Similarresults were obtained using the primers described in Appendix 3. No DNAwas amplified using Primer Pairs 3 and 4 for Chromosome 1 and 7 from thered blood cells of one European patient who was HIV-positive for a yearor less. This suggests that in some Caucasians that the accumulation ofthis human DNA in the red blood cell fraction occurs late afterinfection and possibly under the selective pressure of antiretroviraltreatment. However, amplified DNA was detected in this patient using thePrimer Pairs 1 and 2 shown in Appendix 2, but the amplified DNA bandswere weaker than those for chronically-infected HIV-positive patients.

TABLE 1 Cultured cells Caucasian (HL60) African RBC: HL60 + RBC: HIV+RBC HIV+ treated extract treated with from with antibiotics HIV+antibiotics for 3 subject RBC: RBC: WBC: for 3 RBC: RBC: WBC: months (0No (0 vs 3 HIV− HIV+ HIV+ months HIV− HIV+ HIV+ vs 3 mos) extract days)App.2 Pair 1 rare 100% 0% ↓ 0% 100% 0% ↓ − ↑ Pair 2 rare 100% 0% ↓ 0%100% 0% ↓ − ↑ Chr 1 Pair 1 + + − + − + or −* + − Chr 7 Pair 1 + + − +− + or −* + − + in chronically infected and treated patients − inrecently infected patients

DNA Amplified Using Primer Pairs 1 and 2

MSP2 Primer Pairs 1 and 2 were used to perform PCR on red blood cells ofHIV-positive subjects are removal of white blood cells and other bloodcomponents by Ficoll gradient separation. Primer Pairs 1 and 2 are shownbelow.

Primer Pair 1 18 mer (SEQ ID NO. 3) 5′ GCCTA CAGAT TAAAG GCT 20 mer (SEQID NO. 4) 5′ ATCAT ARTCA CCATC ACCTA Primer Pair 2: 17 mer (SEQ ID NO.5) 5′ CYTAC AGAGT GAAGG CT 20 mer (SEQ ID NO. 6) 5′ ATCAT ARTCA CCATCACCTA

The DNA bands amplified by PCR using Primer Pairs 1 and 2 were 100%homologous with human sequences (primer sequences excluded) present indata-banks for human genomic sequences, respectively in human chromosome1 (clone RPII-332J14 GI:22024579, clone RP11-410C4 GI:17985906, andBuild GRCh37.p5 Primary Assembly-) and in human chromosome 7 (PAC cloneRP4-728H9 GI:3980548; human Build GRCh37.p5, and alternate assemblyHuRefSCAF_1103279188381:28934993-35424761).

Human Chromosome 1 and 7 DNA Sequences Described in Appendix 5

Appendix 5 shows the identities of human chromosome 1 and Chromosome 7sequences that are amplified by primers described in Appendix 3. Primersequences are underlined.

Primer Pair 3: Primer “hChr1114179308 S” upstream of, and“hChr1/14179853 AS” encompassing one end of the 237 bp amplicon relatedto chromosome 1 (546-bp long amplicon) were used to perform PCR onmaterial from red blood cells isolated from other blood components byFicoll gradient.

Primer Pair 4: Primers “hChr7/4292976 S” upstream of, and “hChr7/4294619AS” downstream of the 213 bp amplicon related to chromosome 7 (1,643-bplong amplicon); and the primers described by Appendix 3.

Example 2. Method for Detecting Risk of Acquiring HIV Infection orOpportunistic Infection Associated with HIV Infection or Risk of theProgression of an HIV Infection or Opportunistic Infection

Blood is collected from a subject in the presence of EDTA as ananticoagulant. The red blood cells in the sample are separated frombuffy coat and plasma components of blood using a Ficoll-Hypaquegradient according to the manufacturer's current protocol. DNA in thered blood cell sample is prepared and amplified using a QIAGEN® FastCycling PCR Kit or Taq PCR Core Kit (as described in the current QIAGEN®product catalog) using MSP2 primer pair 1:

(SEQ ID NO: 3) 5′ GCCTA CAGAT TAAAG GCT and (SEQ ID NO: 4) 5′ ATCATARTCA CCATC ACCTA

(SEQ ID NO: 5) 5′ CYTAC AGAGT GAAGG CT and (SEQ ID NO: 6) 5′ATCAT ARTCA CCATC ACCTA.

Amplified DNA is resolved by gel electrophoresis and detected bystaining with ethidium bromide. A subject is classified as being at ahigher risk for acquiring HIV or HIV-associated opportunistic infectionor for when amplified DNA is detected.

FIGS. 1 and 2 were produced on different dates. Lane numbers 1 and 2 areduplicates (from HIV-negative source) and lanes 3 and 4 are duplicates(from HIV-positive source). All DNA samples were extracted from a thirdpassage HL60 cells exposed to an agent originating from red blood cellsof HIV-negative or HIV-positive patients. These panels represent gelelectrophoresis pictures of amplicons obtained by 70 cycles of PCR usingprimers derived from the Ehrlichia MSP2 gene (213 bp) and primersderived from the 16s ribosomal gene of Anaplasma (690 bp). The bands onthe left sides of FIGS. 1(A) and 1(B) show the 213 bp fragment mapped tohuman chromosome 7 amplified by the Ehrlichia MSP2 primers. The bands onthe right sides of FIGS. 1 and 2 show the 690 bp fragment amplified bythe Anaplasma primers (SEQ ID NOS: 24 and 25).

Example 3. Method for Detecting Microorganism Associated with Risk orProgression of HIV Infection or Opportunistic Infection Associated withInfection with HIV

Blood is collected from a subject in the presence of EDTA as ananticoagulant. The red blood cells in the sample are separated frombuffy coat and plasma components of blood using a Ficoll-Hypaquegradient according to the manufacturer's current protocol. DNA in thered blood cell sample is prepared and amplified using a QIAGEN® FastCycling PCR Kit or Taq PCR Core Kit (as described in the current QIAGEN®product catalog) using the primer pair

(SEQ ID NO: 24) 5′-CTG ACG ACA GCC ATG CA and (SEQ ID NO: 25) 5′-GCA GTGGGG AAT ATT GGA CA.

Amplified DNA is resolved by gel electrophoresis and detected bystaining with ethidium bromide. A subject is identified as beinginfected with a microorganism when amplified DNA is detected.

APPENDIX 1 Primers were designed to based on include a conserved 16Srickettsiales16S region of DNA which ribosomal DNA Primers designedbased recognizes on Rickettsiales Rickettsiales and also 16s ribosomalDNA gene SEQ ID NO: Propionobacter 5′-GCAACGCGAAAAACCTTACC SEQ ID NO: 1Rick16S 929S (20 mer) Tm = 45.0° C. 5′-GACGGGCAGTGTGTACAA SEQ ID NO: 2Rick 16S 1373AS (18 mer) Tm = 45.0° C.

APPENDIX 2 MSP2 Primers SEQ ID NO: MSP primer pair 15′-GCCTACAGATTAAAGGCT SEQ ID NO: 3 18-mer 5′-ATCATARTCACCATCACCTA SEQ IDNO: 4 20-mer MSP primer pair 2 5′-CYTACAGAGTGAAGGCT SEQ ID NO: 5 17-mer5′-ATCATARTCACCATCACCTA SEQ ID NO: 6 20-mer

APPENDIX 3 Human chromosome 1 and 7 primers Chromosome 1 primers SEQ IDNO: Primer #1 5′-CCTTACACTCAGCCAGACAT SEQ ID NO: 7 hChr1/14179308 SPrimer #2 5′-CCAGGTGTGTGGCTTATACA SEQ ID NO: 8 hChr1/14179853 AS Primer#3 5′-CATAGCTTCCTAGTAAGTAGACCAG SEQ ID NO: 9 hChr1/14180006 S Primer #45′-AGGGGAGTCTGAGATGGT SEQ ID NO: 10 hChr1/14180401 AS Primer #55′-ACTGGAGAGGTGGAGGTT SEQ ID NO: 11 hChr1/14181093 AS Primer #65′-GGTAATTCCTATGTGCGAGGT SEQ ID NO: 12 hChr1/14181390 AS Primer #75′-TCAAAAGACAGTGGTGACTCT SEQ ID NO: 13 hChr1/14177990 S Primer #85′-ATGTCTGGCTGAGTGTAAGG SEQ ID NO: 14 hChr1/14179327 AS Chromosome 7primers SEQ ID NO: Primer #1 5′-ATGTAGTTGAGCAGTTTTGAATGA SEQ ID NO: 15hChr7/4292976 S Primer #2 5′-TCCTGCCTTAGTGAGGATCT SEQ ID NO: 16hChr7/4293490 S Primer #3 5′-ATGAATAGGTGATGGTGATGACT SEQ ID NO: 17hChr7/4293941 AS Primer #4 5′-CCGTCATTTAAGCCTTTAATCTCA SEQ ID NO: 18hChr7/4294102 S Primer #5 5′-GTAGTCTTTTGGCATCTCTTTGTA SEQ ID NO: 19hChr7/4294619 AS Primer #6 5′-CAGCCTGGAGAACAGAGTG SEQ ID NO: 20hChr7/4294983 AS Primer #7 5′-GATCTAGCAGTTCATAGGAAGGAA SEQ ID NO: 21hChr7/4295251 AS Primer #8 5′-GGCTGAAACAATGGGGTTTT SEQ ID NO: 22hChr7/4291579 S Primer #9 5′-TTTAGTAGACCCCTCGACCA SEQ ID NO: 23hChr7/4293175 AS

APPENDIX 4 Anaplasma 16s rDNA based primers SEQ ID NO: Primer5′-CTGACGACAGCCATGCA SEQ ID NO: 24 Sense Primer 5′-GCAGTGGGGAATATTGG SEQID NO: 25 antisense ACA

APPENDIX 5

The human chromosome 1 sequence amplified by the MSP2 primers shownbelow:

primer identifiers Sequence MSP2 Ac/mMSP2-1019S 5′-CYTACAGAGTGAAGGCTprimer #3) (SEQ ID NO: 5) MSP2 Ac/mMSP2-1128AS 5-ATCATARTCACCATCACCTAprimer #5) (SEQ ID NO: 6) Y = T or C; R = G or ASequence of the 237 bp amplicon (SEQ ID NO: 26) generated with these twoprimers by PCR:

5′-ATCATAGTCA CCATCACCTA CCAGCTGTAT AAGCCACACA CCTGGGAGTC CTCCTAGCCTTTTTCCTCCT CCTCTCATCC TCCATATCCC ATTGACCGTC AGGGCCTACT GAGTCTACACTCCAATTTTC TTTTAAATCT ATCCCCACTG CCACTGTCCT AGTCTAAGGC AATACCATCTGGTCACCCAG ATCATTCCAT AGCTTCCTAG TAAGTAGACC AGCCTTCACT CTGTAAG-3′

underlined nucleotides: primer sequence.

bold nucleotides: divergent nucleotides between MSP2 primers and thecorresponding homologous human Chr 1 sequence.

The human chromosome 7 sequence amplified by the MSP2 primers shownbelow:

Primer identifiers Sequence MSP2 AphMSP2-1019S 5′-GCCTACAGATTAAAGGCTprimer #2) (SEQ ID NO: 3) MSP2 Ac/mMSP2- 5′-ATCATARTCACCATCACCTA primer#5) 1128AS (SEQ ID NO: 6) Y = T or C; R = G or ASequence of the 213 bp amplicon (SEQ ID NO: 27) generated with these twoprimers by PCR:

5′-GCCTACAGAT TAAAGGCTTA AATGACGGTG AAAACTTAGT ATTCTTTGGG TGGACAATAGTGAAATTTGC ACTTTGGACA GAATGACATG TACAAAAAGA GTCAAGAAAC TTTTTAATCTATTTAAAGGA CTCAAAGTAA TTTGTGAAGG CCATAGCGTA AAATAACTTC AGTGGATGGAATGGGATGAT GAATAGGTGA TGGTGACTAT GAT-3′

underlined nucleotides: primer sequence.

bold nucleotides: divergent nucleotides between MSP2 primers and thecorresponding homologous human Chr 7 sequence.

Identities of the human sequences amplified by the human chromosome 1primers or human chromosome 7 primers described in sections A) and B)respectively below.

The sequences described below, except for the primers MSP2-derivedamplicons, are corresponding to human genetic sequences available inNCBI genome databanks (www.ncbi.nlm.nih.gov/projects/genome).

A) Genomic human Chromosome 1 primers #1 (hChr1/14179308 S) and #2(hChr1/14179853 AS) PCR-amplified 546 bp amplicon (SEQ ID NO: 28):

identifier: Amplicon hChr1/14179308-14179853

5′-CCTTACACTC AGCCAGACAT ATATTTGTGT TTTGTTATCC ATGTGCACAG AGACTTTGGCATTCTGGGTG AAGGAAGAAA GAAGAGAATA TACATGGAAA CCCAGGGGTA AGAGAAAAGGACAACAGAGA ATGTGGCATG GGGAATGCTC TGCTGGGTCA CATTGAATGG TTCTGAACCACTGTGGAAAA AAAGGAGTTA GAAAGAATCA GATGCCGAAG GAGCCAATTT TCACAATACTCCGAGACTCA GGGCAAAAGC AGCCTTGTTC TAGTAGCCTA TGGGTAAAAG AAGACACAGAACTGAGGGGA GGACTTTTCC CCTGAGTCCA CCACAAACCG CCATGGAGCT GAGGCAGCCTGAAGTCTCAG GGGCATGGGA GGGATTTGCC TTTTGGATTT CTCCAATGGG ATGTCTTACAGGCACTTCAT ATTTAGCAGA TCCAAAACTT AACTCAGATA CTCCTCTTGC CATATCTGTTCCTCTTGCTG TGTTCCTGAC CATGATTATC ACCATCACCTACCAGCTGTA TAAGCCACAC ACCTGG-3′

underlined nucleotides: hChr1 genomic primer sequence.

bold nucleotides: homologous extremity of the 237 bp amplicon obtainedwith the primers MSP2#3-5.

B) Genomic human Chromosome 7 primers #1 (hChr7/4292976 S) and #5(hChr7/4294619 AS) PCR-amplified 1,644 bp amplicon (SEQ ID NO: 29):

Identifier: Amplicon hChr7/4292976-4294619

5′-ATGTAGTTGA GCAGTTTTGA ATGAGTTTCT TAATCCTGAG TTCTAGTTTA AGAAAATATTAAAAATAAAA AATTATGTCA CCAACTAAAT TTTTACTGCA GATAATCATA AGTTGGTTAGATTGGACCTT CATTGTGAAA TGCAGTAACT TTGGTTTAAG CAATATCCAA AACCAGAAATTGGTCGAGGG GTCTACTAAA TTCCGTTTTC TTTTGTTCTA AACAATTAAA CATTCTAAAATTTAGGGAAA AGGACCAATG GTGCAAACAT TTTAGAGCTG ACAGTTGTGT GCCATATGCCATGATTCTGT TACAAATGAA CAGTATTCAG ATTCAAAATC AGTGTAAACA CTGTGTGTGTGTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTATTTTACA CAGCCATTTA AATATTAACCGCCTTTGAGT ATTAGGGGAA AAAACAGAAA CTAAAAGCGA ATATATTTGT TCCTGAATCCTCCCACCAAA CCACTTTTTA AATTAATTAT ATTTCCTGCC TTAGTGAGGA TCTTCCTATTCATCAAAGAT AAAATACCAA ATATAATTTA CTCTCCTTCT CTACCTCACC CCCAATATTCAACAATTCCC TATTTTATTT TGATTTTACT TCCTATTGTC TCTCAGTGCA TCCTTACTACTGGTTTCTGG CCTCAATGTC TCTTTCCATA AATACTTCCT CCAGGGCTTC AGCAGTGTATATTGCAATCC ATGAGTGTGA TGCCCTTATA AGCTGTACAG GCACAACCCA GGCAAACATACACAATGACC ATAATCATAA CAGTCATTAC TGGTGCCTTT ACTCTGGTTT TATCCATCCCCCAACAATCC TTTAATCCTC CACTAGAGTT TATTCTTTTA AGTGAAAATC TGGATTCTTATCTCCCCTAG TATGTATCTC TTAGTGAATT TTTATATGAG ATAGTCATCA CCATCACCTATTCATCATCC CATTCCATCC ACTGAAGTTA TTTTACGCTA TGGCCTTCAC AAATTTACTTTGAGTCCTTT AAATAGATTA AAAAGTTTCT TGACTCTTTT TGTACATGTC ATTCTGTCCAAAGTGCAAAT TTCACTATTG TCCACCCAAA GAATACTAAG TTTTCACCGT CATTTAAGCCTTTAATCTCA GGATCTCACA ATAAATACAA TCACTCTTTC CTATATGCCT AATCTTCTGCTTGAGCATAA TTTTAATGTG CTAACTATTC TGTAATTATA TATATATTTT TAATTCAGCCACTTCTCTCA CTAGAAAGTG AGTTTGTTGA AATCAGGGTA AGTATATTTT ATGTTTGGATGAATTCCCCA TCACAATACT TCACATGTAG TTATCTAGTC ACCAATTTTT ATTGAAATTAATTGTACATA TAATAAAACT TTAATATAAA ATGTTTCTCT TGAGGGGAGA TTTTCTTTGTAAAACTATCC TTCTGAGCTT TGTGATGTGA TGATTGTCTA ATGTCTGTTG CAAGATTAAGGAAAATGTAT TTGAATGCAA ATGAACTTAC ACTGTCATAC CAAAAGTGTG ATAATTTCTTGCTCCTGAAC TCACTCTCCC TACCTGCCTA TTAAAATCAG AATACACAGA TCTGTATCTGTACAAAGAGA TGCCAAAAGA CTAC-3′

underlined nucleotides: hChr7 genomic primer sequence.

bold nucleotides: homologous extremity of the 213 bp amplicon obtainedwith the primers MSP2#3-5.

The internal underlined nucleotides correspond to the sequence of primerhChr7/4293490 S (hChr7#2).

Other amplicons obtained from HIV positive or HIV-negative patients orfrom both using the human chromosome 1 or human chromosome 7 primersdescribed in sections C) and D) below.

C) Genomic human Chromosome 1 primers #3 (hChr1/14180006 S) and #4(hChr1/14180401 AS) PCR-amplified 396 bp amplicon (SEQ ID NO: 30):

Identifier: Amplicon hChr1/14180006-141a0401

5′-CATAGCTTCC TAGTAAGTAG ACCAGCCTTC AGTCTGAGCC CTCCTCGGTC CTTCCTCCCCAGTGCTGCTG GAGTAATCCT TCTAACACAA CAATGAAAGC AGGTCACTGC GGCTCAAATGATGTCAGCGG CTTTATCATC CATGTTGCCT GGCTTTTCAC AGGCATGTCT TGCAGTGCAGCCTTATAACT CTCTCAACAC AACTCTGTAT CCTCCTCATT CTTCATGCTT TTATAATGTCAAGCCATGTG ACACTCCCTA AATATACCAT GTTTTCTCTT TTTCCTCCTC CCCCTCTCTCATTTGCAGCT TCCCATACTT ATCTTCCTAA ACACTACTCT TTTTGAAATG TTTATTTCAAGGGTTTCTTA TCTTTTAAAC CATCTCAGAC TCCCCT-3′

underlined nucleotide: primer sequences.

bold nucleotide: homologous extremity of the 237 bp amplicon obtainedwith primers MSP2#3-5.

D) Genomic human Chromosome 1 primers #3 (hChr1/14180006 S) and #5(hChr1/14181093 AS) PCR-amplified 1,088 bp amplicon (SEQ ID NO: 31):

Identifier: Amplicon hChr1/14180006-14181093

5′-CATAGCTTCC TAGTAAGTAG ACCAGCCTTC AGTCTGAGCC CTCCTCGGTC CTTCCTCCCCAGTGCTGCTG GAGTAATCCT TCTAACACAA CAATGAAAGC AGGTCACTGC GGCTCAAATGATGTCAGCGG CTTTATCATC CATGTTGCCT GGCTTTTCAC AGGCATGTCT TGCAGTGCAGCCTTATAACT CTCTCAACAC AACTCTGTAT CCTCCTCATT CTTCATGCTT TTATAATGTCAAGCCATGTG ACACTCCCTA AATATACCAT GTTTTCTCTT TTTCCTCCTC CCCCTCTCTCATTTGCAGCT TCCCATACTT ATCTTCCTAA ACACTACTCT TTTTGAAATG TTTATTTCAAGGGTTTCTTA TCTTTTAAAC CATCTCAGAC TCCCCTGGGG ATTACCCCTT TTCCTATGTTTTTATTGTAG CATCCTCACA AATTCACTTT AGTTCCTTCG CATTCTGGTG TCGCTATATATTAGTGGGAC TATGTCCCCA TTAACCTGTT AGATCTCTTG AGAAAAGGGA CATGTCTTTTCATCTTGAGT TCCCCAATAC TTAGTATTGT GCTTAGCATA TGCTAGGTGC TCAGTAAATATTTGATATGT GTGTGAACGA ATGAATCAAT CAATCAATAA CAAATGACAG ACAAACTCCAACCCCCAAAC CTAAAAAAAA AAAATCCAAA CTTTCCCCTT GCTCTTAGTG TAGATACTGCTCATCAACAT AAGGCAAATT CTTCCTGCGC GTCTCAATAC AGAGGAGGCG AGAACTCACAGAATCACAGA ATTAGAGCAC TGGCTTTGGC ATGAGAACAC CCTGAGTTAA AATCTGGCTTCTGCTATTTA TTAGCCACAT GACAGTGAAT CTCCTTGAGC TTCTGTTTTG TACAAACTTAAGTTTGGCTT TGTGATCTTA TTCCTCTTTG GTGCATCTGT ACAACCCAAC TGCTTATTCATATGACACTG CTAAAACATG CCTTGCCTTC TCCCCCACTT TTTTTTTTGG AGACAGAATCTCCCTTTGTC ACCCAGGCTG GAATTCAGTG GCGTGATCTC GGCTCACTGC AACCTCCACCTCTCCAGT-3′

underlined nucleotides: primer sequences.

Bold nucleotides: homologousd extremity of the 237 bp amplicon obtainedwith the primers MSP2#3-5.

The sequence of the 396 bp amplicon hChr1/14180006-14180401 (C) isincluded in the larger size amplicon 1,088 AmpliconhChr1/14180006-14181093.

The internal underlined nucleotides correspond to the reverse-complementsequence of primer hChr1/14180401 AS (hChr1#4).

E) Genomic human Chromosome 7 primers #2 (hChr7/4293490 S) and #6(hChr7/4294983 AS) PCR-Amplified 1,494 bp amplicon (SEQ ID NO: 32):

Identifier: Amplicon hChr7/4293490-4294983

5′-TCCTGCCTTA GTGAGGATCT TCCTATTCAT CAAAGATAAA ATACCAAATA TAATTTACTCTCCTTCTCTA CCTCACCCCC AATATTCAAC AATTCCCTAT TTTATTTTGA TTTTACTTCCTATTGTCTCT CAGTGCATCC TTACTACTGG TTTCTGGCCT CAATGTCTCT TTCCATAAATACTTCCTCCA GGGCTTCAGC AGTGTATATT GCAATCCATG AGTGTGATGC CCTTATAAGCTGTACAGGCA CAACCCAGGC AAACATACAC AATGACCATA ATCATAACAG TCATTACTGGTGCCTTTACT CTGGTTTTAT CCATCCCCCA ACAATCCTTT AATCCTCCAC TAGAGTTTATTCTTTTAAGT GAAAATCTGG ATTCTTATCT CCCCTAGTAT GTATCTCTTA GTGAATTTTTATATGAGATA GTCATCACCA TCACCTATTC ATCATCCCAT TCCATCCACT GAAGTTATTTTACGCTATGG CCTTCACAAA TTACTTTGAG TCCTTTAAAT AGATTAAAAA GTTTCTTGACTCTTTTTGTA CATGTCATTC TGTCCAAAGT GCAAATTTCA CTATTGTCCA CCCAAAGAATACTAAGTTTT CACCGTCATT TAAGCCTTTA ATCTCAGGAT CTCACAATAA ATACAATCACTCTTTCCTAT ATGCCTAATC TTCTGCTTGA GCATAATTTT AATGTGCTAA CTATTCTGTAATTATATATA TATTTTTAAT TCAGCCACTT CTCTCACTAG AAAGTGAGTT TGTTGAAATCAGGGTAAGTA TATTTTATGT TTGGATGAAT TCCCCATCAC AATACTTCAC ATGTAGTTATCTAGTCACCA ATTTTTATTG AAATTAATTG TACATATAAT AAAACTTTAA TATAAAATGTTTCTCTTGAG GGGAGATTTT CTTTGTAAAA CTATCCTTCT GAGCTTTGTG ATGTGATGATTGTCTAATGT CTGTTGCAAG ATTAAGGAAA ATGTATTTGA ATGCAAATGA ACTTACACTGTCATACCAAA AGTGTGATAA TTTCTTGCTC CTGAACTCAC TCTCCCTACC TGCCTATTAAAATCAGAATA CACAGATCTG TATCTGTACA AAGAGATGCC AAAAGACTAC TTTCATGCTGCAACATGATT ATGTGCCCCC AAAACCTGGA TATTTATAGT ATAGTATCCA GTATTTTCAATCTAAGCTGT ACTGGAGCCC GAAGCTAAAG GAAAATTAGT AATACTGATG CTCCCTTTATTTAAACTTTT AAGACTTTAT CATGGCATTA ATTTTGACTT TTAAAAATAT TATCATTTTTTTTGGACCCC CTTAAATTTT GTTCCCGAGT TGAATGCCTC ACTGGGACTT GGGTGAATGAATGCTCACCC TAGTCCTAGA TTAGGTACTC ATCTTAAATA CTGTTAGTTT GGGGTGGTTTTTTTTTTTTT TTTTTTTTTT TTTTTGACAG AGCCTCACTC TGTTCTCCAG GCTG-3′

underlined nucleotides: hChr7 genomic primer sequence.

bold nucleotides: homologous sequence of the 213 bp amplicon obtainedwith the primers MSP2#2-5.

A part of the sequence 1,644 bp amplicon hChr7/4292976-4294619 (B) isincluded in the amplicon 1,494 bp Amplicon hChr7/4293490-4294983 (E).The internal underlined nucleotides correspond to the reverse-complementsequence of primer hChr7/4294619 AS (hChr7#5).

APPENDIX 6

An amplicon of the 16s rDNA primers (SEQ ID NOS: 24 and 25) is shownbelow. The amplified DNA originated from the red blood cells of anHIV-negative subject passaged in HL60 cells. Similar DNA is amplifiedfrom samples originating from red 5 blood cells of HIV-positivesubjects.

Amplicon (SEQ ID NO: 33) from HIV-negative subject obtained usingprimers (SEQ ID NOS: 24 and 25):

>TS6-EMK-4-HIV-_Anae#2-5 wo/ primers seq.=681 bp

5′-GCACCTGTATGTGAATTCCCGAAGGCACTCCCGCATCTCTGCAGGATTCTCACTATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCTACTTATCGCGTTAACTGCGCCACTAAAGTCTCAAGGACCCCAACGGCTAGTAGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAATCTCAGTGTCAATATTATGCCAGGAAGCTGCCTTCGCCATCGGCATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACTTCCCTCTCACATATTCTAGCACCACCAGTATCACATGCAGTTCCCAGGTTAAGCCCGGGGATTTCACATGTGACTTAATGAGCCACCTACACTCGCTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGCAGGTAACGTCTAATCTAATGGGTATTAACCATTAGCCTCTCCTCCCTGCTTAAAGTGCTTTACAACCAAAAGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTGCCCCCCATT-3′

What is claimed is:
 1. A primer for detecting an agent that isassociated with human red blood cells, selected from the groupconsisting of SEQ ID NOS: 3-23 or a primer effective for use inamplifying at least twenty consecutive nucleotides of the same DNA assaid primers SEQ ID NOS: 3-23.
 2. The primer according to claim 1,comprising a pair of primers comprising a sense primer and an antisenseprimer selected from the group consisting of (SEQ ID NOS: 3 and 4) and(SEQ ID NOS: 5 and 6) or a pair of primers effective for use inamplifying at least twenty consecutive nucleotides of the same DNA assaid primers selected from the group consisting of (SEQ ID NOS: 3 and 4)and (SEQ ID NOS: 5 and 6).
 3. The primer according to claim 1,comprising a pair of primers comprising a sense primer and an antisenseprimer selected from the group consisting of (SEQ ID NOS: 7-14) and (SEQID NOS: 15-23) or a pair of primers effective for use in amplifying atleast twenty consecutive nucleotides of the same DNA as said primersselected from the group consisting of (SEQ ID NOS: 7-14) and (SEQ IDNOS: 15-23).
 4. Isolated or purified DNA having a DNA sequence and a DNAlength that is produced by amplifying DNA using a polymerase chainreaction, using a pair of primers selected from the group consisting of:(i) primers described in Appendix 2 selected from the group consistingof pair 1 (SEQ ID NOS: 3 and 4) and pair 2 (SEQ ID NOS: 5 and 6); and(ii) primers described by Appendix 3 comprising a sense primer and anantisense primer selected from the group consisting of (SEQ ID NOS: 7-14or SEQ ID NOS: 15-23).
 5. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 26).
 6. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 27).
 7. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 28).
 8. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 29).
 9. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 30).
 10. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 31).
 11. The isolated or purified DNA of claim 4, thatcomprises a DNA sequence or fragment thereof described by Appendix 5(SEQ ID NO: 32).
 12. The isolated or purified DNA of claim 4, containedwithin a vector that self-replicates in live cells.
 13. A method fordetecting an agent that is associated with human red blood cells,comprising: performing a polymerase chain reaction amplification of DNAfrom the human red blood cells using at least one primer selected fromthe group consisting of SEQ ID NOS: 3-25 or a primer effective for usein amplifying at least twenty consecutive nucleotides of the same DNA assaid primers SEQ ID NOS: 3-25; and detecting a selectively producedamplicon from the polymerase chain reaction amplification.
 14. Themethod according to claim 13, wherein the at least one primer comprisesa pair of primers selected from the group consisting of (SEQ ID NOS: 3and 4) and (SEQ ID NOS: 5 and 6) or a pair of primers effective for usein amplifying at least twenty consecutive nucleotides of the same DNA assaid primers selected from the group consisting of (SEQ ID NOS: 3 and 4)and (SEQ ID NOS: 5 and 6).
 15. The method according to claim 14, whereinthe at least one primer comprises a pair of primers selected from thegroup consisting of (SEQ ID NOS: 7-14) and (SEQ ID NOS: 15-23) or a pairof primers effective for use in amplifying at least twenty consecutivenucleotides of the same DNA as said primers selected from the groupconsisting of (SEQ ID NOS: 7-14) and (SEQ ID NOS: 15-23).
 16. The methodaccording to claim 15, wherein the at least one primer comprises a pairof primers (SEQ ID NOS: 24 and 25) or a pair of primers effective foruse in amplifying at least twenty consecutive nucleotides of the sameDNA as said primers (SEQ ID NOS: 24 and 25).
 17. The method according toclaim 15, wherein the selectively produced amplicon comprises a DNAsequence or fragment thereof described by Appendix 5 (SEQ ID NOS:26-32).
 18. The method according to claim 15, wherein the selectivelyproduced amplicon comprises a DNA sequence or fragment thereof describedby Appendix 6 (SEQ ID NO: 33).
 19. A method for detecting an agentassociated with mature anucleated human red blood cells, comprising:extracting DNA from a sample of cells exposed to the agent associatedwith mature anucleated human red blood cells from a patient; performinga polymerase chain reaction amplification of extracted DNA, using atleast one primer selected from the group consisting of SEQ ID NOS: 3-25or a primer effective for use in amplifying at least twenty consecutivenucleotides of the same DNA as said primers SEQ ID NOS: 3-25; anddetecting a selectively produced amplicon from the polymerase chainreaction amplification.
 20. The method according to claim 19, whereinthe selectively produced amplicon comprises a DNA sequence or fragmentthereof described by Appendix 5 or Appendix 6 (SEQ ID NOS: 26-33).